Inflammatory conditions in companion animals: Inflammation is usually a normal, healthy response to injury or infection, but sometimes the inflammatory response is disproportionate or abnormal, so that the inflammation, rather than promoting healing, seriously damages normal tissues, resulting in chronic pain, contributing to a wide variety of serious disorders, and in some cases even causing death. Inflammatory bowel disease (IBD), for example, is a debilitating and progressive disease involving inflammation of the gastrointestinal tract. Symptoms include abdominal pain, cramping, diarrhea and bleeding.
One indication of such inflammatory diseases is the presence of inflammatory cells such as neutrophils and macrophages at local sites of inflammation. Inflammation is a response of vascularized tissue to infection and/or injury and it is affected by adhesion of leukocytes to the endothelial cells of blood vessels and their infiltration into the surrounding tissues. Such local concentrations can be detected by invasive methods requiring biopsy procedures and pathology analysis. The inflammatory state can also be systemic, i.e. polypeptides secreted by inflammatory cells become detectable in the blood serum.
Inflammatory bowel disease (IBD) describes idiopathic gastrointestinal disorders characterized by persistent or recurrent gastrointestinal (GI) signs and histological evidence of GI inflammation for which no underlying cause can be found. Effective treatment of IBD requires differentiating the condition from other gastrointestinal disorders that do not necessarily involve chronic inflammation. While certain diagnostics have been developed for humans, these diagnostics are not always accurate, and the lack of accurate diagnostics is even more acute for companion animals such as dogs and cats Inflammatory bowel disease is typically identified by veterinarians as being the most common cause of intestinal disease in companion animals, but accurate data on its prevalence is lacking mainly because diagnosis of the disease is challenging. The disease varies greatly, not only in severity but also in its anatomical distribution throughout the GI of companion animals, particularly lower GI, and perhaps most importantly the type of inflammatory reaction involved. Lymphoplasmacytic enteritis (LPE) is the most common form reported in companion animals followed by eosinophilic gastroenteritis (EGE) that is less common, and granulomatous enteritis (GE) that is rare.
Canine and feline IBD bear little resemblance clinically or histologically to the human IBD forms (Xavier and Podolsky, Nature, 448:427-434 (2007); Cerquetella et al., World J. Gastroent., 16: 1050-1056 (2010). IBD and IBD-related conditions are likely to affect a significant portion of companion animals during their lifetime, and when left untreated, leads to an increase of morbidity, deteriorating quality of life and in some cases cancer. Prompt and accurate diagnosis of the condition is likely to result on rapid and adequate interventions leading to significant improvement in the quality of life of companion animals.
The most common clinical signs in companion animal IBD are vomiting and diarrhea, although the disease can present broader clinical signs including but not limited to abdominal pain, altered appetite and weight loss, bloating and cramping, and even flatulence. All these clinical symptoms are general in nature and overlap with many other potential conditions making definitive diagnosis extremely difficult. The difficulty in diagnosing IBD and differentiating from other superficially similar conditions such as irritable bowel syndrome (IBS), food sensitivities and/or gastrointestinal infections hampers early and effective treatment.
The best current methodology to diagnose IBD in companion animals requires using relatively costly, labor-intensive and intrusive clinical, radiographic, endoscopic, and/or histological techniques. And despite all these techniques, there is a high degree of subjectivity. Histopathological assessment of gastrointestinal biopsies remain the gold standard to diagnose GI inflammation in companion animals. However, the quality of specimens may vary, agreement between pathologists might be lacking, and differentiation between tissues affected by different conditions may be difficult requiring significant intervention, cost and time. The term IBD includes cases in which histological evidence of inflammation is found without obvious underlying cause, and all other etiologies have been excluded. Overall, the diagnosis of IBD remains a process of exclusion, and thus, represents a lengthy and costly process, which also contributes to unnecessary companion animal suffering and morbidity.
Inflammatory bowel disease (IBD) in companion animals poses a major challenge to veterinarians. The initial symptoms are often confused with non-IBD acute or chronic bowel conditions, particularly for those veterinarians unfamiliar with the disease. Therefore, IBD remains undiagnosed and untreated. In the worst case scenario, IBD is misdiagnosed and companion animals are potentially receiving inadequate medical care resulting in increased morbidity and associated companion animal owner discomfort.
Only a few immunological and inflammatory markers have been validated for use in companion animals. There is therefore a need for markers that are both sensitive and specific for companion animals with chronic inflammatory diseases, such as IBD. The availability of rapid and less intrusive methods to diagnose IBD tailored specifically for companion animals would represent a major clinical advance in veterinary medicine and would facilitate earlier and more appropriate therapeutic intervention to treat diseased companion animals. There is a need for a more effective, less intrusive diagnostic method tailored specifically for companion animals to rule out inflammation related IBD if it is not the underlying condition or to rule in IBD.
A further challenge is distinguishing among inflammatory conditions, food sensitivities, and other gastrointestinal disorders.
Food sensitivities in companion animals: Food sensitivity is a unique disorder that is defined as an adverse reaction to specific foods that may cause severe illness. Food sensitivity, in its chronic form, is an immunologic sensitivity to a food involving T and B lymphocytes as part of the chronic cell mediated immune system (also called delayed or type IV hypersensitivity) that may include noticeable symptoms such as abdominal pain, diarrhea, constipation, and weight loss, and may also include less noticeable effects due to malabsorption of fluids and dietary nutrients, such as osteoporosis, anemia, or vitamin deficiencies. Without proper diagnostic testing, which currently is limited to invasive means such as endoscopic intestinal biopsy and the presence of symptoms which can be confounded with other gastrointestinal related diseases, the subject may unknowingly continue to consume the food triggering the chain of reactions which may have long term health implications.
One such immunologic food sensitivity is gluten sensitivity where subjects display an immunologic reaction to dietary gluten contained in wheat, barley, rye, and oats, that results in any degree of intestinal histopathology. The gluten-induced immunologic process causes villous atrophy and inflammation of the small intestine, in turn, resulting in diarrhea and weight loss from malabsorption of fluids, electrolytes, and dietary nutrients. Despite the fact that chronic diarrhea is the most common symptom of food sensitivity to gluten in its classic, villous atrophic, form, there have been no studies of the prevalence of gluten related sensitivity or other food sensitivities in companion animals presenting to veterinarians with chronic diarrhea or other common gastrointestinal symptoms. Additionally, there are no adequate methods in the prior art to diagnose for food sensitivities when the patient presents with little or no common gastrointestinal symptoms or when the symptoms are also attributable to other diseases. This frequently results in either no diagnosis or misdiagnosis of a food sensitivity.
In addition to the gastrointestinal issues triggered by food sensitivities, they have also been associated with other diverse conditions in humans such as dermatopathies (e.g. dermatitis herpetiformis) and neurological disorders such as gluten ataxia. In cases of gluten ataxia, although less than 10% show gastrointestinal signs of disease, over 34% will have evidence of enteropathy on biopsy.
Elimination diets in which controlled proteins and carbohydrates are used as sources remain the main approach used by veterinarians to diagnose food sensitivities. The elimination diet approach has significant challenges though. These diets have to be maintained for at least 12 weeks to be effective with significant compliance issues. Further, because there is no current robust methodology to determine the specific source of sensitivity, veterinarians favor the introduction of pan-hypoallergenic diets with highly hydrolyzed proteins which are costly and lack clear determination of which ingredient or ingredients are causing the sensitivity.
Food sensitivity in some cases can be defined as an intolerance to proteins including storage proteins from grains such as wheat and corn. The food sensitivity is characterized by a chronic inflammatory state of the proximal small intestinal mucosa that heals when foods containing prolamins rich proteins are excluded from the diet and returns when these foods are reintroduced. Complex adaptive and innate immune reactions result in chronic inflammation of the mucosa and result in various structural and functional changes. There is atrophy of the small intestinal villi, deepening of the crypts, and infiltration of the lamina propria and intraepithelial compartments with chronic inflammatory cells. The functional changes include decreased digestion of food, decreased absorption of macronutrients and micronutrients, and increased net secretion of water and solute. Although the mucosal damage is primarily cellular, untreated food sensitivity is also associated with a humoral immune response that consists of both secreted intestinal and circulating serologic antibodies directed against the reticulin and endomysium of connective tissue such as endomysial antibodies, and against various peptides derived predominantly from wheat, “anti-gliadin antibodies” and from corn “anti-zein antibodies”.
Prolamins are a group of proteins soluble in 40-70% ethanol and are the major storage proteins of all cereal grains. Prolamins are further classified in sub-families based on differences in aqueous solubility and ability to form disulfide interactions. Most prolamins share common structural features including the presence of amino acid sequences consisting of repeated blocks based on one or more short peptide motifs, or enriched in specific amino acid residues. These features are responsible for the high proportions of glutamine, proline and other specific amino acids (e.g. histidine, glycine, methionine, and phenylalanine).
The gluten proteins of wheat consist of an approximately equal amount mixture of monomeric gliadins and polymeric glutenin subunits forming 80% of the total storage protein content in the wheat kernel. The remainder is albumins (12%) and globulins (8%). The highly seed storage proteins of wheat and barley are classified on the basis of their solubility into water-soluble albumins, salt-soluble globulins, alcohol-soluble (40%-70%) gliadins, and alcohol-insoluble glutenins. In wheat, the alcohol insoluble fraction of prolamins is comprised of the High Molecular Weight (HMW) glutenin subunits (650-850 residues long) and the Low Molecular Weight (LMW) glutenin subunits (270-330 residues long), whereas the alcohol soluble fraction is comprised of gliadins (250-720 residues long) further separated into sulphur-poor w-gliadins and the sulphur-rich α/β and γ gliadins. In barley the homologous proteins are named D-, C-, B- and γ-hordeins, respectively. Glutamine (Gln or Q) and proline (Pro or P) are two major amino acids that comprise 35 and 15% of the gluten proteins, respectively, and as such are also classified prolamins.
The prolamins of maize, called zeins, and of other panicoid cereals such as sorghum and millets are comprised of one major group of proteins (α-zeins) rich in glutamine, proline, alanine, and leucine residues accounting for 70% of the total zein fraction and several minor groups (β, γ, δ-zeins). Amino acid sequence comparisons demonstrate that the β, γ and δ-zeins are all members of the prolamin superfamily, but only the γ-zeins contain repeated amino acid sequences (either two or eight tandem repeats of Pro-Pro-Pro-Val-His-Leu). The β-zeins and δ-zeins are both rich in methionine. The α-zeins contain only one or two cysteine residues per molecule and are present in the grain as monomers or oligomers, while the β-, γ- and δ-zeins are all richer in cysteine and form polymers.
The amino acid composition of the prolamin polypeptides makes them highly resistant to gastric and pancreatic proteases. The partially digested polypeptides move from the stomach into the intestine, where they are incompletely hydrolyzed by the exopeptidases of the brush border membrane. The immunogenicity of these peptides may be intensified by deamidation of selected glutamine residues by tissue transglutaminase 2 (tTG2). These polypeptide complexes may trigger mucosal inflammation and loss of absorptive surface area manifested by a broad spectrum of symptoms and nutritional deficiencies in subjects with active food sensitivity.
Alpha-Amylase inhibitors also present in grains of the cereal family are composed of 120-160 amino-acid residues forming five disulfide bonds. These inhibitors may cause upon repeated exposure harmful effects.
Transglutaminase 2 is a multifunctional enzyme that belongs to transglutaminases which catalyze the crosslinking of proteins by epsilon-(gamma-glutamyl) lysine isopeptide bonds. Tissue transglutaminase 2 (tTG2 or TTG2), identified as an endomysial autoantigens, is an intestinal digestive enzyme which deamidates already partially digested dietary gluten e.g. gliadin peptides. In food sensitivity predisposed subjects, tTG2 triggers autoimmune responses that are characterized by the production of tTG2 antibodies. Epidermal transglutaminase (eTG 3 or TTG3) is another member of the transglutaminase family that can also function as an autoantigen that is manifested by a skin rash clinically known as dermatitis herpetiformis (DH).
Food sensitivity in companion animals poses a major challenge to veterinarians. The most common clinical signs in pet food sensitivity are vomiting and diarrhea, although the disease can present broader clinical signs including but not limited to abdominal pain, altered appetite and weight loss, bloating and cramping, and even flatulence. All these clinical symptoms are general in nature and overlap with many other potential conditions making definitive diagnosis extremely difficult. The difficulty in differentially diagnosing food sensitivity and differentiate from other similar conditions such as inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and/or gastrointestinal infections hampers early and effective treatment of these diseases.
The best current methodology to diagnose food sensitivity in companion animals either requires using relatively costly, labor-intensive and intrusive clinical, radiographic, endoscopic, and/or histological techniques and/or the implementation of diet elimination trials followed by the re-introduction of specific ingredients to confirm the food sensitivity. The term food sensitivity includes cases in which histological evidence of inflammation is found without obvious underlying cause, and all other etiologies have been excluded. Overall, the diagnosis of food sensitivity remains a process of exclusion, and thus, represents a lengthy and costly process that also contributes to unnecessary pet suffering and morbidity.
Veterinarians need rapid, accurate and relatively non-intrusive methods to detect, distinguish and diagnose chronic food sensitivity, inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), and/or gastrointestinal infections, which may present with similar symptoms but which require very different therapeutic interventions. Despite the significant and long-felt need for effective diagnostic methods tailored specifically for companion animals, the tools to differentially diagnose these conditions in companion animals remain unavailable, hampering the early and effective treatment of these diseases.
The present invention addresses these needs and provides related advantages as well.